The protein NfxB, involved in conferring resistance to quinolones in Pseudomonas aeruginosa, has a helixturn-helix motif which is similar to that of other DNA-binding proteins. It appears to affect the membraneassociated energy-driven efflux of some antibiotics (H. Nikaido, Science 264:382-388, 1994). We constructed a plasmid that overproduced NfxB in Escherichia coli and purified the protein. Two species of NfxB (23 and 21 kDa), which are probably translated from different initiation codons, were isolated. Both proteins are also expressed in vivo in P. aeruginosa, with the 23-kDa NfxB being the major species. NfxB specifically binds upstream of the nfxB coding region as demonstrated by gel retardation and DNase I footprinting. Expression of the ⌽(nfxB-lacZ ؉ )(Hyb) gene was repressed in the presence of the nfxB gene product provided by a second compatible plasmid in E. coli. In the P. aeruginosa wild-type strain (PAO2142), NfxB was undetectable by immunoblotting; however, it was detected in the nfxB missense mutant (PK1013E). These results suggested that NfxB negatively autoregulates the expression of nfxB itself. Since the 54-kDa outer membrane protein (OprJ) (N. Masuda, E. Sakagawa, and S. Ohya, Antimicrob. Agents Chemother. 39:645-649, 1995) was overproduced in nfxB mutants, NfxB may also regulate the expression of membrane proteins that are involved in the drug efflux machinery of P. aeruginosa.The intrinsic resistance of Pseudomonas aeruginosa to a large variety of antimicrobial agents was shown to be due mainly to efflux system effects and partly to the low-permeability outer membrane (10, 14).The P. aeruginosa nfxB mutants, which show a 16-fold increase in resistance to norfloxacin, were isolated spontaneously (7). The mutants also show hypersusceptibility to ␤-lactam and aminoglycoside antibiotics (17). Some nfxB mutants overproduce the 54-kDa outer membrane protein, OprJ (11), little of which is produced in the wild-type strain (7,17). Antibiotic resistance in the nfxB mutants is probably not due to altered outer membrane permeability but to multidrug efflux pump effects (14, 18).The nfxB gene cloned from the wild type as well as a mutant (the nfx13E mutation) has been sequenced (16,17). The amino acid sequence of NfxB revealed that it has a helix-turn-helix motif that might be responsible for its ability to bind in a sequence-specific manner to DNA, and it has no significant hydrophobic membrane-spanning regions (16). nfx13E is a missense mutation which replaces an arginine residue in the putative helix-turn-helix domain with a glycine residue in the mutant (16). The wild-type nfxB gene can restore susceptibility to norfloxacin in P. aeruginosa with the nfx13E mutation. This evidence suggests that NfxB binds to DNA and regulates the expression of genes that encode the outer membrane protein(s). It is possible that Nfx13E mutant protein is unable to bind to DNA and thus loses its regulatory function. In addition, nfxB expression might be autoregulated by NfxB protein itself. Like the LysR-type transcri...

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